bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438099; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Susceptibility of rosaceous fruits and apple cultivars to postharvest 2 rot by Paecilomyces niveus 3 4 Tristan W. Wang* and Kathie T. Hodge 5 6 Section of Plant Pathology and Plant-Microbe Biology, School of Integrative Plant Science, 7 Cornell University, Ithaca, NY 14853 8 9 *Corresponding author 10 Email: [email protected] 11 Abstract 12 Paecilomyces rot of apples is a postharvest disease caused by Paecilomyces niveus, a 13 problematic spoiling agent of fruit juices and derivatives. The fungus produces ascospores that 14 can survive food processing and germinate in finished fruit products. Processing apple fruits 15 infected with Paecilomyces rot can lead to P. niveus contaminated juices. Because the fungus 16 produces the mycotoxin patulin, juice spoilage by P. niveus is an important health hazard. Little 17 is known about the disease biology and control mechanisms of this recently described 18 postharvest disease. Following Koch’s postulates, we determined that a range of previously 19 untested rosaceous fruits and popular apple cultivars are susceptible to Paecilomyces rot 20 infection. We also observed that two closely related food spoiling fungi, Paecilomyces fulvus 21 and Paecilomyces variotti, were unable to infect, cause symptoms in, or reproduce in wounded 22 fruits. Our results highlight the unique abilities of Paecilomyces niveus to infect a variety of 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438099; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 fruits, produce patulin, and form highly-resistant spores capable of spoiling normally shelf- 24 stable products. 25 Keywords: Paecilomyces rot, postharvest, Rosaceae, Paecilomyces niveus, patulin 26 27 Introduction 28 Patulin is a serious mycotoxin that can cause gastrointestinal, immunological, and neurological 29 damage, and is an important contaminant of apple products and their derivatives (Puel et al. 30 2010). One 2009 survey detected patulin in 23% of apple juices and ciders sampled from 31 Michigan retail grocery stores, with 11.3% of these samples containing patulin over the FDA 32 limit of 50ppb (Harris et al. 2009). Contamination by this mycotoxin is thought to occur through 33 postharvest fruit infection and food spoilage by patulin-producing microbes. At least 18 34 Penicillium, Aspergillus, and Paecilomyces species are known to produce patulin (Puel et al. 35 2010) and the mycotoxin has been detected in a wide variety of other fruit products including 36 pear, peach, cherry, apricot, orange, and mango juices and jams (Erdogan et al. 2018, Hussain 37 et al. 2020, Moake et al. 2005, Spadaro et al. 2008). 38 One important patulin producer, Paecilomyces niveus Stolk & Samson (Byssochlamys nivea 39 Westling), uniquely threatens juice production as it is a thermotolerant mold that creates 40 durable ascospores. The ascospores are the predominant structures responsible for P. niveus 41 food contamination. Contamination by P. niveus is a long-standing issue and the fungus has 42 been found to contaminate a wide variety of fruit products including apple-based products, 43 concentrated orange juice, strawberry puree, and tomato paste (Kotzekidou 1997, dos Santos 44 et al. 2018). 45 Paecilomyces niveus is a common soil fungus, found present in a third of New York orchard soil 46 samples (Biango-Daniels and Hodge 2018). It has been thought that P. niveus contamination of 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438099; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 47 food products is environmental, originating from soil, air, and equipment. Even small quantities 48 of this homothallic fungus pose a hazard for juice production as a single P. niveus spore can give 49 rise to heat-resistant ascospores. Recently, P. niveus was identified as the causal agent of 50 Paecilomyces rot, a postharvest disease of apples (Biango-Daniels and Hodge 2018) and citrus 51 fruits (Wang and Hodge 2020). Biango-Daniels et al. (2019) showed that processing apple fruits 52 infected with Paecilomyces rot can result in apple juice significantly contaminated with patulin 53 and viable P. niveus ascospores. Since the description of Paecilomyces rot (Biango-Daniels and 54 Hodge 2018), Paecilomyces niveus has been found infecting apples in a fruit market in China 55 (Khokhar et al. 2019). In addition, the fungus was observed growing on peas in Serbia (Dragic et 56 al. 2016) and on aphids in Brazil (Zawadneak et al. 2015). 57 The broad range of fruit products in which P. niveus and patulin have been found led us to 58 hypothesize that the fungus may be able to infect and reproduce in a range of fruits. In 59 addition, we sought to further characterize the disease biology of Paecilomyces rot by testing 60 the susceptibility of other popular apple cultivars (Empire, Fuji, Granny Smith, and Golden 61 Delicious). Lastly, we asked whether pathogenesis in our wound challenges was unique to P. 62 niveus. Like P. niveus, two closely related heat-resistant molds known to contaminate fruit 63 products, Paecilomyces fulvus and Paecilomyces variotii, produce heat-resistant ascospores 64 capable of surviving temperatures above 85˚C (Houbraken et al. 2006, Samson et al. 2009). We 65 hypothesized that both can also cause symptoms, reproduce in apple fruits, and contaminate 66 apple products via infected apple fruits. 67 To test the preceding hypotheses, we tested the wound-infecting ability of P. niveus in fruits of 68 a variety of apple relatives: peaches, pears, sweet cherries and sour cherries. In addition, we 69 inoculated and compared lesion development in four popular apple cultivars. Lastly, to test for 70 the disease-causing abilities of P. niveus relatives in wounded apple fruits, we inoculated apple 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438099; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 71 fruits with Paecilomyces variotii and Paecilomyces fulvus and observed them for symptom 72 development. 73 74 Materials and methods 75 Fruit 76 Detached peach fruits (Prunus persica, cv. Lori Anne) (n=26) and pear fruits (Pyrus communis, 77 cv. Green D’Anjou) (n=31) were selected at a local supermarket based on their uniformity in 78 size, and absence of both wounds and disease symptoms. Sour cherries (Prunus cerasus, cv. 79 Montmorency) (n=40) and sweet cherries (Prunus avium, cv. Kristin) (n=31) were freshly picked 80 from a local New York orchard. For apple cultivar susceptibility testing, Empire (n=31), Fuji 81 (n=28), Golden Delicious (n=31), and Granny Smith (n=31) apples were purchased from a local 82 supermarket. These four cultivars were chosen based on their popularity in US markets. Three 83 of these cultivars (Empire, Fuji, and Granny Smith) have not previously been tested for 84 susceptibility to Paecilomyces rot. For susceptibility tests involving P. fulvus and P. variotii, 30 85 Empire apples were used for inoculation of each fungus. 86 87 Fungal pathogens 88 Paecilomyces niveus strain CO7, isolated from culled apple fruits in New York, was used to 89 inoculate fruits using the method described in Biango-Daniels and Hodge (2018). We have 90 sequenced the full genome of this strain (Biango-Daniels et al. 2018), and found that sequences 91 from ITS and BenA regions can reliably be used to confirm identity as P. niveus. P. niveus strain 92 MC4, isolated from New York residential garden soil and P. niveus strain 106-3, isolated from 93 New York orchard soil, were also used in fruit inoculation and identified using their ITS and 94 BenA regions. Paecilomyces fulvus strain 7, obtained from the Worobo lab collection and 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438099; this version posted April 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 95 originally isolated from spoiled food and Paecilomyces variotii strain 103-2, isolated from NY 96 soil, were identified morphologically and by sequencing of the ITS region. 97 To produce inoculum, fungi were grown from 5mm plugs taken from the edge of 2-week-old 98 colonies on PDA. Five plugs of P. niveus were cultured on PDA. The fungus was allowed to grow 99 in the dark for two weeks at 25˚C, covering tyndallized toothpicks that had been autoclaved 100 twice: once in water and once in potato dextrose broth the following day. Control toothpicks 101 were similarly treated, but in the absence of the fungus. Both P. fulvus and P. variotii were 102 grown as described above to produce toothpick inoculum.